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Molecular encapsulation of citral or d-limonene flavor by spray drying Chisho Yamamotoa, Takeshi Furutab, Tze Loon Neohc, HidefumiYoshiic
a United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Japan
([email protected]) b Department of Biotechnology, Tottori University, Tottori, Japan ([email protected]) c Department of Applied Biological Science, Kagawa University, Takamatsu, Japan
([email protected]) ABSTRACT
Lemongrass oil and d-limonene are typical flavor oils used in the food and cosmetic industries. Lemongrass oil displays some therapeutic properties such as analgesic, antimicrobial, fungicidal, deodorant, etc. Citral, the main active component of lemongrass oil, consists of a mixture of two isomeric acyclic monoterpene aldehydes: geranial and neral. Molecular encapsulation of these flavors was investigated by spray drying. Cyclodextrin (CD) and modified starch were used as encapsulants that made up the wall material of the spray-dried powders. CD can control flavor release rate and protect the encapsulated flavor from volatilization by the formation of inclusion complex. CD content in the wall material affected the morphology, flavor retention, and flavor stability of the spray-dried powders. d-Limonene and citral in lemongrass oil encapsulated within the matrix of CD were the most stable. However, the retention of d-limonene and citral was the highest in their respective wall materials of pure modified starch and highly branched cluster dextrin (HBCD). Microscopy of the spray-dried powders revealed that hollow structure of the powders was influenced by the content of cyclodextrin in the wall material. The inclusion complexation ability of β-CD could possibly remedy the inefficiency of CapsulTM in protecting the encapsulated citral against oxidation over time due to its “leaky” structure. Keywords: Spray drying; cyclodextrin: encapsulation: d-limonene; inclusion INTRODUCTION
Cyclodextrins (CDs) are doughnut shaped cyclic oligosaccharides with an interior cavity and they form specific inclusion complexes with many organic compounds [1]. CDs are made from starch using the CD transglycosylase enzyme to hydrolyze and cyclize the starch to form closed circular molecules or CDs. Typically, these CD molecules contain six, seven or eight glucose molecules and are called α-, β-, or γ-CD. These glucose units are linked by α-1, 4 bonding found in starch. The hydrogen and glucosidic oxygen atoms face toward the inside of the CD macro-ring and form an electron-dense or apolar lining in the cavity, which can interact with hydrophobic compounds that match the CD cavity to form an association or complex. On the other hand, the polar hydroxyl groups of the glucose monomers face toward the outside of the macro-ring and are responsible for the aqueous-solubility of the CDs and their complexes. In food related applications, flavor compounds are being encapsulated into CDs for better retention and protection from various possible means of deterioration, as well as for controlled delivery. Molecular inclusion of flavors has been used as a method of converting liquid flavors into a dry form. For food applications of flavor powders, controlled release properties are needed for many food products such as microwave entrees, snacks and desserts, etc. Encapsulation is generally carried out in commercial practice by spray drying because it handles both water-soluble and oil-soluble flavor systems equally well. Spray drying is the most commonly used technique for the production of dry flavorings [2]. Shiga et al. [3] investigated the powdery encapsulation of shiitake flavors extracted from dried shiitake with CD and maltodextrin by spray drying. The flavor retentions were markedly increased by use of α-CD and maltodextrin in combination as the encapsulant. Liu et al. [4] investigated molecular encapsulation of l-menthol in CD to prevent the loss of the hydrophobic flavor compound during the drying of single droplet. They proposed a simple mathematical model for estimating the flavor retention. The theoretical results by their model could well estimate the final retention of l-menthol encapsulated in the blends of β-CD and maltodextrin. Reineccius et al. [5] studied three commonly used flavor industry solvents (propylene glycol, triacetin, and triethyl citrate) for their capacity to interfere with the ability of α-, β-, and γ-CD to form molecular inclusion complexes with flavors. However, there are few investigations about the effect of the preparation method of
inclusion complex between flavor and CD on the inclusion ratio or retention of flavor in spray-dried powders. There are no study to our knowledge about the effect of mixture wall materials such as modified starch or maltodextrin and CDs on the retention of flavor and morphologies of spray-dried powders. In this study, spray drying was employed to prepare flavor encapsulated powders using natural CDs. The flavor was encapsulated within the powder particles via the combined mode of encapsulation: (i) inclusion by natural CDs and (ii) encapsulation by modified starch or highly-branched cyclic dextrose (HBCD). MATERIALS & METHODS
Preparation of the inclusion complex powders d-Limonene was selected as a model flavor for encapsulation into natural CDs, namely α-, β-, and γ-CD. In encapsulation by CDs alone, 10 to 30 wt% CD solutions were mixed with 1.5 molar-folds of d-limonene. To investigate the effect of inclusion time, the mixtures were incubated at room temperature for 0, 1 and 24 h, followed by the homogenization and eventually pulverization using an Ohkawara L8 spray dryer (Ohkawara Kakouki Co., Ltd., Yokohama, Japan). The drying conditions of the spray dryer were, inlet temperature: 120, 160, 200 °C; rotation speed of atomizer: 30,000 rpm; feed rate: 30 mL/min; and air flow rate: 130 kg/h. The effect of mixture wall material on the encapsulation of d-limonene was investigated with α-CD and HBCD. The percentage of α-CD in the mixture wall material was changed from 0 to 100 wt% at interval of 20 wt% and the solid content of the feed liquid was 20 wt%. The amount of d-limonene added to all the feed liquids was identical, regardless of their α-CD to HBCD ratios. Meanwhile, the molecular encapsulation of citral in lemongrass oil was investigated by using the mixture wall materials of β-CD and modified starch, CAPSUL®. Citral was added at a molar amount equivalent to that of β-CD in the feed liquid with 100 wt% β-CD as the wall material. The same amount of citral was added to the other feed liquids, regardless of their β-CD to CAPSUL® ratios. Flavor quantification Solvent extraction was used for quantification of flavors in the spray-dried powders. Briefly, 0.1 g of the spray-dried powder was added with 4 mL of water and 1 mL of cyclohexanone-containing chloroform in a test tube which was then sealed with a silicone-lined screw cap and heated at 90 °C for 20 min. Intermittent vigorous shaking of the test tube was performed during heating. After extraction, the samples were centrifuged at 3000 rpm for 15 min. One microliter of the chloroform phase was analyzed with a gas chromatograph (GC-14A, Shimadzu Corp., Kyoto, Japan) equipped with a PEG-20M packed column (2 m × 3.2 mm i.d.) and a flame ionization detector. Measurement of release time course of encapsulated flavors The release time courses of flavors from the spray-dried powders were measured according to the method reported previously [5]. The spray-dried powders (ca. 0.1 g) were weighed into glass bottles (ϕ 8 mm × 16 mm) and held in an air bath at 50 °C. Humid air was flowed into the glass bottles through silicon tubes at the rate of 40 mL/min to sweep out the flavor released from the powder. At prescribed intervals, the glass bottles were taken out and the residual flavor was measured by gas chromatography (GC) as described above. Flavor stability in mixture wall materials The stability of flavors in the spray-dried powders with the mixture wall materials was measured by the retention of flavor in the powders incubated at 50 °C. The powder was sealed in a closed test tube (ϕ 15 mm × 75 mm). The residual flavor content was measured by GC. Powder morphology The spray-dried powders were analyzed with a scanning electron microscope (JEOL JSM-6060, JEOL Ltd., Tokyo, Japan) for visualization of the external and internal appearance of the particles. Images of the specimen were obtained at an accelerating voltage of 2.0 kV and 500-1,000 × magnifications. RESULTS & DISCUSSION
Inclusion complex powders by spray drying Solid content of feed solutions was varied between 10-30% to determine the effect on the initial inclusion ratio. In the cases of the feed liquids with 100 wt% CD, the spray-dried powders were rather inclusion complexes between the flavors and CDs than flavor encapsulated powders. Hence, the flavor retention would be more appropriately described as inclusion ratio which was defined as the molar ratio of flavor compounds to CDs. Although a little difference in the inclusion ratio was observable with the increase of solid content, the inclusion ratios of d-limonene in both the β- and γ-CD complex powders were almost independent of the CD concentration. The SEM images for the inclusion complex powders are illustrated in Figure 1. The shape
of the particles was different depending on both the type of CD and the incubation time. The inclusion complex powders of α- and β-CD spray-dried from the 24-h-incubated feed liquids are comprised of rod- and cube-shaped crystals, respectively, whereas shorter incubation times resulted in larger spherical particles. For γ-CD, all the particles were spherical independent of the incubation time. Figure 2 shows the effect of incubation time on the X-ray diffractogram of the spray-dried inclusion complex powders. Peak intensities of the powders from α- and β-CD increased with incubation time. However, the trend was not observed in the powders from γ-CD. This might be due to the larger cavity size of γ-CD than those of α- and β-CD, which influence the facilily of complex formation between d-limonene and γ-CD. The release of d-limonene from these spray-dried inclusion complex powders was investigated at 50 °C, 75% relative humidity (RH). The release characteristics are dependent on the type of CD and the incubation time. d-Limonene included in α-CD was hard to release. Only 10% of the initial amount could be released in 150 h. On the other hand, for β- and γ-CD, the release was faster for powders prepared from unincubated feed liquids than for that from the incubated ones. Avrami’s equation was applied to the release time courses of the complexed d-limonene in CDs. The release rate constants of the powders from the unincubated feed liquids were 5.3 × 10-9 h-1, 4.8 × 10-3 h-1, and 4.7 × 10-3 h-1 for α-, β-, and γ-CD, respectively, while for the samples from the 24-h-incubated feed liquids were 3.8 × 10-8 h-1, 2.3 × 10-4 h-1, and 2.5 × 10-3 h-1 for the respective CDs. The release rate constants are affected by the crystal state of the particular inclusion complex. The results suggest that the incubation time of the feed liquid is an important factor dictating the storage stability of the complexed flavors. The incubation time affected significantly the morphology of the spray-dried inclusion complex powders and as well the release characteristics of the complexed flavors.
Figure 1. SEM micrographs of spray-dried inclusion complex powders. Solid content of CD in feed liquid: 10 wt%, Inlet air temperature: 160 °C, and incubation time: 0, 1, and 24 h.
Figure 2. Effect of incubation time of feed liquid on the X-ray diffractograms of the spray-dried inclusion complex powders of natural CDs. Asterisks indicate the specific peaks of the inclusion complexes.
Inte
nsity
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α‐CD β‐CD γ‐CD
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*
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5 10 15 20 5 10 15 20 5 10 15 20２θ （degree)
Mixture wall materials of α-CD and HBCD in encapsulation of d-limonene Effect of mixture wall material between α-CD and HBCD on the morphology of the spray-dried powders and the retention of d-limonene was investigated. Figure 3 shows the surface morphology of the spray-dried powders with the mixture wall materials of α-CD and HBCD. By addition of HBCD, the crystal particles of α-CD were seemed to be engulfed within the HBCD matrix. The inner structure of spray-dried powders was observed by splitting particles with adhesive tapes. As shown in Figure 4, the crystals of α-CD were vividly observable on the inside of the particles with wall materials containing 40-100 wt% α-CD. In the formation of pharmaceutical powders with crystalline compounds, the morphology of the spray dried powder is very important in controlling the powder properties. Crystal habit is important because particle shape affects the physical properties such as solubility. High drying rates during spray drying may plasticize the feed liquids, impeding the formation of nuclei crystals that can serve as nucleation sites for crystal growth during storage. Vehring [6] suggested in his review on the recent developments in the area of particle engineering via spray drying that particle morphology was an important factor influencing the physical characteristics of spray-dried powders.
Figure 3. SEM micrographs of spray-dried powders with the mixrure wall materials of α-CD and HBCD. The percentage of α-CD was changed from 0 to 100 wt% at an interval of 20 wt%.
Figure 4. Internal structures of spray-dried powders with the mixture wall materials of α-CD and HBCD. The percentage of α-CD was changed from 0 to 100 wt% at an interval of 20 wt%.
X1000 10µm ×1000 10µm ×1000 10µm
0 % 20 % 40 %
60 % 80 % 100 %
×1000 10µm ×1000 10µm ×1000 10µm
X5000 5µm X2000 10µm X 3000 5µm
X2000 10µm X2000 10µm X2000 10µm
0 % 20 % 40 %
60 % 80 % 100 %
The retention of d-limonene and the particle size of the spray-dried powders were measured for the mixture wall materials of α-CD and HBCD. Figure 5 shows the effects of α-CD content in the mixture wall materials on d-limonene retention and average particle size. With further increase in α-CD content above 40 wt%, the powder particles developed into increasingly fragile structures which broke into smaller fragments upon particle size determination. HBCD does not have good emulsification ability. However, the retention of d-limonene was the highest, which was about 0.57. The composition of wall material significantly influenced the retention of d-limonene which decreased as the α-CD content increased. The lowest retention was achieved at 60 wt% α-CD. These results suggest that the crystal of α-CD inhibit the film formation on the surface of the atomized droplets during spray drying. By use of high molecular dextrin or sugar as wall material, the dried film could act as a semi-permeable membrane permitting the continued loss of water, but efficiently retaining volatile flavors. The crystal compounds might break down the skin wall during the initial drying. The flavor retention depends on mainly the wall hardening of wall material such as maltodextrin. The film-forming property is one of the known key features that determine the suitability of a wall material for encapsulation. The powder composed of α-CD alone as the wall material was the smallest in particle size, whereas those with of the wall materials containing 40 and 60 wt% α-CD were the largest. The retention of d-limonene in the mixture wall materials of α-CD and HBCD might depend on the inclusion of d-limonene in α-CD and the HBCD matrix that covered the α-CD crystal aggregates. The particle size decreased as the α-CD content increased further. The particle diameter of the powders with mixture wall materials of α-CD and HBCD were smaller than that of typical spray-dried powders with maltodextrin as the wall material. The stability of d-limonene in the spray-dried powders was studied at 50 °C. Figure 6 shows the effect of α-CD content in the mixture wall materials on the stability of d-limonene in the spray-dried powders. Since the retention of d-limonene decreased almost linearly with the incubation time, the oxidation rate of d-limonene Figure 5. Effect of α-CD content in the mixture wall materials with HBCD on the d-limonene retention ( ) and average
particle size ( ).
Figure 6. Effect of α-CD content in the mixture wall materials with HBCD on the stability of d-limonene. The mixture wall materials contain 100 ( ), 80 ( ), 60 ( ), 40 ( ), 20 ( ), and 0 wt% ( ) α-CD. The spray-dried powders were
incubated in sealed bottles at 50 °C under dry condition.
d-Li
mon
ene
rete
ntio
n (-)
α-CD/(α-CD+HBCD) (%)
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er (μ
m)
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ene
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α-CD content (%)0 20 40 60 80 100
0
0.01
0.02
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Oxi
datio
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te c
onst
ant
k (1
/s)
could be estimated by zero-order reaction. The oxidation kinetic constant k (day-1) decreased linearly with the content of α-CD in the mixture wall material, as shown in the inset of Figure 6. [ ]( )CDα11007.4 2 −−×= −k (1) where [α-CD] is the mass fraction of α-CD in the mixture wall material. The stability of d-limonene remarkably depended on the content of α-CD in the mixture wall materials. This might indicate that the complexed d-limonene in α-CD was stable and HBCD did not affect its stability because α-CD crystals inhibit the skin formation. Mixture wall materials of β-CD and modified starch for encapsulation of lemongrass oil The spray drying condition was the same as that use for the preparation of the d-limonene encapsulated powders with the mixture wall materials of α-CD and HBCD. The difference in average particle diameter of the powders was insignificant despite the difference in viscosity of the feed liquids. The retention of citral decreased slightly with the increase in β-CD content from 0 to 40 wt%. The retention ability of CapsulTM was then observed to reduce enormously from 40 to 60 wt% β-CD, causing the citral retention to reach the minimum. However, the effect of inclusion complexation by β-CD plausibly gradually became noticeable above 60 wt% β-CD, which was reflected by the proportional increase in citral retention with increasing β-CD content. The retention of citral at different β-CD contents was similar to that of d-limonene in the mixture wall materials of α-CD and HBCD. The oxidation stability of citral was investigated at 50 °C. The oxidation rate of citral could be correlated with Avrami’s equation at the mechanism parameter of 0.5. Analogous to the trend demonstrated by d-limonene encapsulated in the mixture wall materials of α-CD and HBCD, citral complexed with pure β-CD was the most stable. CONCLUSION
The molecular inclusion of flavor compounds into CDs is one of the techniques in flavor encapsulation. The length of incubation time exhibited noticeable effects on the crystallinity and release behavior of the spray-dried inclusion complex powders between d-limonene and α-CD. The stability of d-limonene encapsulated within the mixture wall materials of α-CD and HBCD depended on the α-CD content. The morphology of the spray-dried powders with the mixture wall materials was significantly influenced by the CD content. The evolution of flavor retention with CD content was similar in both the mixture wall materials of α-CD and HBCD and β-CD and CAPSUL®. REFERENCES
[1] Szejtli J. 1988.Cyclodextrins in Food. In: Cyclodextrin Technology. Kluwer Academic Publishers, Dordrecht, The Netherlands.
[2] Anker M.H. & Reineccius G.A. 1988. Encapsulated Orange Oil – Influence of Spray-dryer Air Temperatures on Retention and Shelf Life. In: Risch S.J. & Reineccius G.A. (Eds). Flavor Encapsulation. American Chemical Society, Washington, DC, USA.
[3] Shiga H., Yoshii H., Ohe H., Yasuda M., Furuta T., Kuwahara H., Ohkawara M., & Linko P. 2004. Encapsulation of Shiitake (Lenthinus edodes) Flavors by Spray Drying. Bioscience, Biotechnology, and Biochemistry, 68(1), 66-71.
[4] Liu X.-D., Furuta T., Yoshii H., Linko P. & Coumans W.J. 2000. Cyclodextrin Encapsulation to Prevent the Loss of l- Menthol and Its Retention During Drying. Bioscience, Biotechnology, and Biochemistry, 64(8), 1608–1613.
[5] Reineccius T.A., Reineccius G.A., & Peppard T.L. 2003. Flavor Release from Cyclodextrin Complexes: Comparison of α, β, and γ Types. Journal of Food Science, 68(4), 1234-1239.
[6] Vehring R. 2008. Pharmaceutical Particle Engineering via Spray Drying. Pharmaceutical Research, 25(5), 999-1019.
